Anti-cd38 human antibodies and uses thereof

ABSTRACT

The present invention provides recombinant antigen-binding regions and antibodies and functional fragments containing such antigen-binding regions that are specific for CD38, which plays an integral role in various disorders or conditions. These antibodies, accordingly, can be used to treat, for example, hematological malignancies such as multiple myeloma. Antibodies of the invention also can be used in the diagnostics field, as well as for investigating the role of CD38 in the progression of disorders associated with malignancies. The invention also provides nucleic acid sequences encoding the foregoing antibodies, vectors containing the same, pharmaceutical compositions and kits with instructions for use. The invention also provides isolated novel epitopes of CD38 and methods of use therefore.

This application is a Continuation of U.S. application Ser. No.14/630,042 filed on Feb. 24, 2015, which is pending, which is aContinuation of U.S. application Ser. No. 13/427,305, filed Mar. 22,2012, which is abandoned, which is a Divisional of U.S. application Ser.No. 10/588,568, which issued as U.S. Pat. No. 8,263,746, which is the USNational Stage application of PCT/IB05/002476, filed Feb. 7, 2005, whichclaims priority to U.S. provisional application numbers 60/541,911 filedFeb. 6, 2004, 60/547,584 filed Feb. 26, 2004, 60/553,948 filed Mar. 18,2004, and 60/599,014 filed Aug. 6, 2004, and 60/614,471, filed Oct. 1,2004, the contents of each of which are incorporated herein in theirentireties.

BACKGROUND OF THE INVENTION

CD38 is a type-II membrane glycoprotein and belongs to the family ofectoenzymes, due to its enzymatic activity as ADP ribosyl-cyclase andcADP-hydrolase. During ontogeny, CD38 appears on CD34+ committed stemcells and lineage-committed progenitors of lymphoid, erythroid andmyeloid cells. It is understood that CD38 expression persists only inthe lymphoid lineage, through the early stages of T- and B-celldevelopment.

The up-regulation of CD38 serves as a marker for lymphocyteactivation—in particular B-cell differentiation along the plasmacytoidpathway. (Co-)receptor functions of CD38 leading to intracellularsignaling or intercellular communication via its ligand, CD31, arepostulated, as well as its role as an intracellular regulator of asecond messenger, cyclic ADPr, in a variety of signaling cascades.However, its physiological importance remains to be elucidated, sinceknock out of the murine analogue or anti-CD38 auto-antibodies in humansdo not appear to be detrimental.

Apart from observing its expression in the hematopoetic system,researchers have noted the up-regulation of CD38 on various cell-linesderived from B-, T-, and myeloid/monocytic tumors, including B- orT-cell acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),Non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). In MM, forexample, strong CD38 expression is witnessed in the majority of allpatient samples.

Hence, over-expression of CD38 on malignant cells provides an attractivetherapeutic target for immunotherapy. Of special attraction is the factthat the most primitive pluripotent stem cells of the hematopoieticsystem are CD38-negative and that the extent of cytotoxic effects byADCC or CDC correlates well with the expression-levels of the respectivetarget.

Current approaches of anti-CD38 therapies can be divided in two groups:in vivo and ex vivo approaches. In in vivo approaches, anti-CD38antibodies are administered to a subject in need of therapy in order tocause the antibody-mediated depletion of CD38-overexpressing malignantcells. Depletion can either be achieved by antibody-mediated ADCC and/orCDC by effector cells, or by using the anti-CD38 antibodies as targetingmoieties for the transport of cytotoxic substances, e.g. saporin, to thetarget cells, and subsequent internalization. In the ex vivo approach,cell population, e.g. bone marrow cells, comprising CD38 overexpressingmalignant cells are removed from an individual in need of treatment andare contacted with anti-CD38 antibodies. The target cells are eitherdestroyed by cytotoxic substances, e.g. saporin, as described for the invivo approach, or are removed by contacting the cell population withimmobilized anti-CD38 antibodies, thus removing CD38 overexpressingtarget cells from the mixture. Thereafter, the depleted cell populationis reinserted into the patient.

Antibodies specific for CD38 can be divided in different groups,depending on various properties. Binding of some antibodies to the CD38molecule (predominantly aa 220-300) can trigger activities within thetarget cell, such as Ca2+ release, cytokine release, phosphorylationevents and growth stimulation based on the respective antibodyspecificity (Konopleva et al., 1998; Ausiello et al., 2000), but noclear correlation between the binding site of the various knownantibodies and their (non-)agonistic properties could be seen (Funaro etal., 1990).

Relatively little is known about the efficacy of published anti-CD38antibodies. What is known is that all known antibodies seem toexclusively recognize epitopes (amino acid residues 220 to 300) locatedin the C-terminal part of CD38. No antibodies are known so far that arespecific for epitopes in the N-terminal part of CD38 distant from theactive site in the primary protein sequence. However, we have found thatOKT10, which has been in clinical testing, has a relatively low affinityand efficacy when analyzed as chimeric construct comprising a human Fcpart. Furthermore, OKT10 is a murine antibody rendering it unsuitablefor human administration. A human anti-CD38 scFv antibody fragment hasrecently been described (WO 02/06347). However, that antibody isspecific for a selectively expressed CD38 epitope.

Correspondingly, in light of the great potential for anti-CD38 antibodytherapy, there is a high need for human anti-CD38 antibodies with highaffinity and with high efficacy in mediating killing of CD38overexpressing malignant cells by ADCC and/or CDC.

The present invention satisfies these and other needs by providing fullyhuman and highly efficacious anti-CD38 antibodies, which are describedbelow.

SUMMARY OF THE INVENTION

It is an object of the invention to provide human and humanizedantibodies that can effectively mediate the killing ofCD38-overexpressing cells.

It is another object of the invention to provide antibodies that aresafe for human administration.

It is also an object of the present invention to provide methods fortreating disease or and/or conditions associated with CD38 up-regulationby using one or more antibodies of the invention. These and otherobjects of the invention are more fully described herein.

In one aspect, the invention provides an isolated antibody or functionalantibody fragment that contains an antigen-binding region that isspecific for an epitope of CD38, where the antibody or functionalfragment thereof is able to mediate killing of a CD38+ target cell (LP-1(DSMZ: ACC41) and RPMI-8226 (ATCC: CCL-155)) by antibody-dependentcellular cytotoxicity (“ADCC”) with an at least two- to five-fold betterefficacy than the chimeric OKT10 antibody having SEQ ID NOS: 23 and 24(under the same or substantially the same conditions), when a human PBMCcell is employed as an effector cell, and when the ratio of effectorcells to target cells is between about 30:1 and about 50:1. Such anantibody or functional fragment thereof may contain an antigen-bindingregion that contains an H-CDR3 region depicted in SEQ ID NO: 5, 6, 7, or8; the antigen-binding region may further include an H-CDR2 regiondepicted in SEQ ID NO: 5, 6, 7, or 8; and the antigen-binding regionalso may contain an H-CDR1 region depicted in SEQ ID NO: 5, 6, 7, or 8.Such a CD38-specific antibody of the invention may contain anantigen-binding region that contains an L-CDR3 region depicted in SEQ IDNO: 13, 14, 15, or 16; the antigen-binding region may further include anL-CDR1 region depicted in SEQ ID NO: 13, 14, 15, or 16; and theantigen-binding region also may contain an L-CDR2 region depicted in SEQID NO: 13, 14, 15, or 16.

In another aspect, the invention provides an isolated antibody orfunctional antibody fragment that contains an antigen-binding regionthat is specific for an epitope of CD38, where the antibody orfunctional fragment thereof is able to mediate killing of aCD38-transfected CHO cell by CDC with an at least two-fold betterefficacy than chimeric OKT10 (SEQ ID NOS: 23 and 24) under the same orsubstantially the same conditions as in the previous paragraph. Anantibody satisfying these criteria may contain an antigen-binding regionthat contains an H-CDR3 region depicted in SEQ ID NO: 5, 6, or 7; theantigen-binding region may further include an H-CDR2 region depicted inSEQ ID NO: 5, 6, or 7; and the antigen-binding region also may containan H-CDR1 region depicted in SEQ ID NO: 5, 6, or 7. Such a CD38-specificantibody of the invention may contain an antigen-binding region thatcontains an L-CDR3 region depicted in SEQ ID NO: 13, 14, or 15; theantigen-binding region may further include an L-CDR1 region depicted inSEQ ID NO: 13, 14, or 15; and the antigen-binding region also maycontain an L-CDR2 region depicted in SEQ ID NO: 13, 14, or 15.

Antibodies (and functional fragments thereof) of the invention maycontain an antigen-binding region that is specific for an epitope ofCD38, which epitope contains one or more amino acid residues of aminoacid residues 43 to 215 of CD38, as depicted by SEQ ID NO: 22. Morespecifically, an epitope to which the antigen-binding region binds maycontain one or more amino acid residues found in one or more of theamino acid stretches taken from the list of amino acid stretches 44-66,82-94, 142-154, 148-164, 158-170, and 192-206. For certain antibodies,the epitope may be linear, whereas for others, it may be conformational(i.e., discontinuous). An antibody or functional fragment thereof havingone or more of these properties may contain an antigen-binding regionthat contains an H-CDR3 region depicted in SEQ ID NO: 5, 6, 7, or 8; theantigen-binding region may further include an H-CDR2 region depicted inSEQ ID NO: 5, 6, 7, or 8; and the antigen-binding region also maycontain an H-CDR1 region depicted in SEQ ID NO: 5, 6, 7, or 8. Such aCD38-specific antibody of the invention may contain an antigen-bindingregion that contains an L-CDR3 region depicted in SEQ ID NO: 13, 14, 15,or 16; the antigen-binding region may further include an L-CDR1 regiondepicted in SEQ ID NO: 13, 14, 15, or 16; and the antigen-binding regionalso may contain an L-CDR2 region depicted in SEQ ID NO: 13, 14, 15, or16.

Peptide variants of the sequences disclosed herein are also embraced bythe present invention. Accordingly, the invention includes anti-CD38antibodies having a heavy chain amino acid sequence with: at least 60percent sequence identity in the CDR regions with the CDR regionsdepicted in SEQ ID NO: 5, 6, 7, or 8; and/or at least 80 percentsequence homology in the CDR regions with the CDR regions depicted inSEQ ID NO: 5, 6, 7, or 8. Further included are anti-CD38 antibodieshaving a light chain amino acid sequence with: at least 60 percentsequence identity in the CDR regions with the CDR regions depicted inSEQ ID NO: 13, 14, 15 or 16; and/or at least 80 percent sequencehomology in the CDR regions with the CDR regions depicted in SEQ ID NO:13, 14, 15 or 16.

An antibody of the invention may be an IgG (e.g., IgG₁), while anantibody fragment may be a Fab or scFv, for example. An inventiveantibody fragment, accordingly, may be, or may contain, anantigen-binding region that behaves in one or more ways as describedherein.

The invention also is related to isolated nucleic acid sequences, eachof which can encode an antigen-binding region of a human antibody orfunctional fragment thereof that is specific for an epitope of CD38.Such a nucleic acid sequence may encode a variable heavy chain of anantibody and include a sequence selected from the group consisting ofSEQ ID NOS: 1, 2, 3, or 4, or a nucleic acid sequence that hybridizesunder high stringency conditions to the complementary strand of SEQ IDNO: 1, 2, 3, or 4. The nucleic acid might encode a variable light chainof an isolated antibody or functional fragment thereof, and may containa sequence selected from the group consisting of SEQ ID NOS: 9, 10, 11,or 12, or a nucleic acid sequence that hybridizes under high stringencyconditions to the complementary strand of SEQ ID NO: 9, 10, 11, or 12.

Nucleic acids of the invention are suitable for recombinant production.Thus, the invention also relates to vectors and host cells containing anucleic acid sequence of the invention.

Compositions of the invention may be used for therapeutic orprophylactic applications. The invention, therefore, includes apharmaceutical composition containing an inventive antibody (orfunctional antibody fragment) and a pharmaceutically acceptable carrieror excipient therefor. In a related aspect, the invention provides amethod for treating a disorder or condition associated with theundesired presence of CD38 or CD38 expressing cells. Such methodcontains the steps of administering to a subject in need thereof aneffective amount of the pharmaceutical composition that contains aninventive antibody as described or contemplated herein.

The invention also relates to isolated epitopes of CD38, either inlinear or conformational form, and their use for the isolation of anantibody or functional fragment thereof, which antibody of antibodyfragment comprises an antigen-binding region that is specific for saidepitope. In this regard, a linear epitope may contain amino acidresidues 192-206, while a conformational epitope may contain one or moreamino acid residues selected from the group consisting of amino acids44-66, 82-94, 142-154, 148-164, 158-170 and 202-224 of CD38. An epitopeof CD38 can be used, for example, for the isolation of antibodies orfunctional fragments thereof (each of which antibodies or antibodyfragments comprises an antigen-binding region that is specific for suchepitope), comprising the steps of contacting said epitope of CD38 withan antibody library and isolating the antibody(ies) or functionalfragment(s) thereof.

In another embodiment, the invention provides an isolated epitope ofCD38, which consists essentially of an amino acid sequence selected fromthe group consisting of amino acids 44-66, 82-94, 142-154, 148-164,158-170, 192-206 and 202-224 of CD38. As used herein, such an epitope“consists essentially of” one of the immediately preceding amino acidsequences plus additional features, provided that the additionalfeatures do not materially affect the basic and novel characteristics ofthe epitope.

In yet another embodiment, the invention provides an isolated epitope ofCD38 that consists of an amino acid sequence selected from the groupconsisting of amino acids 44-66, 82-94, 142-154, 148-164, 158-170,192-206 and 202-224 of CD38.

The invention also provides a kit containing (i) an isolated epitope ofCD38 comprising one or more amino acid stretches taken from the list of44-66, 82-94, 142-154, 148-164, 158-170, 192-206 and 202-224; (ii) anantibody library; and (iii) instructions for using the antibody libraryto isolate one or more members of such library that binds specificallyto such epitope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a provides nucleic acid sequences of various novel antibodyvariable heavy regions.

FIG. 1b provides amino acid sequences of various novel antibody variableheavy regions. CDR regions HCDR1, HCDR2 and HCDR3 are designated from N-to C-terminus in boldface.

FIG. 2a provides nucleic acid sequences of various novel antibodyvariable light regions.

FIG. 2b provides amino acid sequences of various novel antibody variablelight regions. CDR regions LCDR1, LCDR2 and LCDR3 are designated from N-to C-terminus in boldface.

FIG. 3 provides amino acid sequences of variable heavy regions ofvarious consensus-based HuCAL antibody master gene sequences. CDRregions HCDR1, HCDR2 and HCDR3 are designated from N- to C-terminus inboldface.

FIG. 4 provides amino acid sequences of variable light regions ofvarious consensus-based HuCAL antibody master gene sequences. CDRregions LCDR1, LCDR2 and LCDR3 are designated from N- to C-terminus inboldface.

FIG. 5 provides the amino acid sequence of CD38 (SWISS-PROT primaryaccession number P28907).

FIG. 6 provides the nucleotide sequences of the heavy and light chainsof chimeric OKT10.

FIG. 7 provides a schematic overview of epitopes of representativeantibodies of the present invention.

FIG. 8 provides the DNA sequence of pMORPHO h IgG1 1 (bp 601-2100) (SEQID NO: 32): The vector is based on the pcDNA3.1+ vectors (Invitrogen).The amino acid sequence encoded by the DNA sequence is presented (SEQ IDNO: 35), and the amino acid sequence of the NH-stuffer sequence isindicated in bold, whereas the final reading frames of the VH-leadersequence and the constant region gene are printed in non-bold.Restriction sites are indicated above the sequence. The priming sites ofthe sequencing primers are underlined. The antisense strand of the DNAsequence is presented in SEQ ID NO. 44.

FIG. 9 provides the DNA sequence of Ig kappa light chain expressionvector pMORPHO h Igic 1 (bp 601-1400) (SEQ ID NO: 33): The vector isbased on the pcDNA3.1+ vectors (Invitrogen). The amino acid sequencessequence encoded by the DNA sequence is presented (SEQ ID NO: 36), andthe amino acid sequence of the Nx-stuffer sequence is indicated in bold,whereas the final reading frames of the Vic-leader sequence and of theconstant region gene are printed in non-bold. Restriction sites areindicated above the sequence. The priming sites of the sequencingprimers are underlined. The antisense strand of the DNA sequence ispresented in SEQ ID NO. 45.

FIG. 10 provides the DNA sequence of HUCAL® Ig lambda light chain vectorpMORPHO h Igk t (bp 601-1400) (SEQ ID NO: 34): The amino acid sequenceencoded by the DNA sequence is presented (SEQ ID NO: 37), and the aminoacid sequence of the VX-stuffer sequence is indicated in bold, whereasthe final reading frames of the VX-leader sequence and of the constantregion gene are printed in non-bold. Restriction sites are indicatedabove the sequence. The priming sites of the sequencing primers areunderlined. The antisense strand of the DNA sequence is presented in SEQID NO. 46.

FIG. 11 provides the results of the proliferation assay: PBMCs from 6different healthy donors (as indicated by individual dots) were culturedfor 3 days in the presence of HUCAL® antibodies Mab#1 (=MOR03077), Mab#2(=MOR03079), and Mab#3 (=MOR03080), the reference antibody chOKTl0, theagonistic (ag.) control IB4, an irrelevant HUCAL® negative control IgG1(NC) and a murine IgG2a (Iso) as matched isotype control for IB4. Astandard labeling with BrdU was used to measure proliferation activityand its incorporation (as RLU=relative light units) analyzed via achemiluminescence-based ELISA.

FIG. 12 provides the results of the IL-6 Release Assay: PBMCs from 4-8different healthy donors (as indicated by individual dots) were culturedfor 24 hrs in the presence of HUCAL® antibodies Mab#1 (=MOR03077), Mab#2(=MOR03079), and Mab#3 (=MOR03080), the reference antibody chOKTlO, theagonistic (ag.) control IB4, an irrelevant HUCAL® negative control (NC)and medium only (Medium). IL-6 content in relative light units (RLU) wasanalyzed from culture supernatants via a chemiluminescence based ELISA.

FIG. 13 provides data about the cytotoxicity towards CD34+/CD38+progenitor cells: PBMCs from healthy donors harboring autologousCD34+/CD38+ progenitor cells were incubated with HUCAL® Mab#1(=MOR03077), Mab#2 (=MOR03079), and Mab#3 (=MOR03080), the positivecontrol (PC=chOKTlO) and an irrelevant HUCAL® negative control for 4hours, respectively. Afterwards, the cell suspension was mixed withconditioned methyl-cellulose medium and incubated for 2 weeks. Colonyforming units (CFU) derived from erythroid burst forming units (BFU-E;panel B) and granulocyte/erythroid/macrophage/megakaryocyte stem cells(CFU-GEMM; panels B) and granulocyte/macrophage stem cells (CFU-GM;panel C) were counted and normalized against the medium control(“none”=medium). Panel A represents the total number of CFU (Total CFUc)for all progenitors. Mean values from at least 10 different PBMC donorsare given. Error bars represent standard error of the mean.

FIG. 14 provides data about ADCC with different cell-lines:

-   -   a: Single measurements (except for RPMI8226: average from 4        indiv. Assays); E:T-ratio: 30:1    -   b: Namba et al., 1989    -   c: 5 μg/ml used for antibody conc. (except for Raji with 0.1        μg/ml)    -   d: addition of retinoic assay for stimulation of CD38-expression        specific killing [%]=[(exp. killing−medium killing)/(1−medium        killing)]*100    -   PC: Positive control (=chOKT10)    -   MM: Multiple myeloma    -   CLL: Chronic B-cell leukemia    -   ALL: Acute lymphoblastic leukemia    -   AML: Acute myeloid leukemia    -   DSMZ: Deutsche Sammlung für Mikroorganismen and Zellkulturen        GmbH    -   ATCC: American type culture collection    -   ECACC: European collection of cell cultures    -   MFI: Mean fluorescence intensities.

FIG. 15 provides data about ADCC with MM-samples:

-   -   ^(a): 2-4 individual analyses

FIG. 16 provides the experimental results of mean tumor volumes aftertreatment of human myeloma xenograft with MOR03080: group 1: vehicle;group 2: MOR03080 as hIgG1 1 mg/kg 32-68 days every second day; group 3:MOR03080 as hIgG1 5 mg/kg 32-68 days every second day; group 4: MOR03080as chIgG2a 5 mg/kg 32-68 days every second day; group 5: MOR03080 ashIgG1 1 mg/kg, 14-36 days every second day; group 6: untreated

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novel antibodies thatare specific to or have a high affinity for CD38 and can deliver atherapeutic benefit to a subject. The antibodies of the invention, whichmay be human or humanized, can be used in many contexts, which are morefully described herein.

A “human” antibody or functional human antibody fragment is herebydefined as one that is not chimeric (e.g., not “humanized”) and not from(either in whole or in part) a non-human species. A human antibody orfunctional antibody fragment can be derived from a human or can be asynthetic human antibody. A “synthetic human antibody” is defined hereinas an antibody having a sequence derived, in whole or in part, in silicofrom synthetic sequences that are based on the analysis of known humanantibody sequences. In silico design of a human antibody sequence orfragment thereof can be achieved, for example, by analyzing a databaseof human antibody or antibody fragment sequences and devising apolypeptide sequence utilizing the data obtained therefrom. Anotherexample of a human antibody or functional antibody fragment, is one thatis encoded by a nucleic acid isolated from a library of antibodysequences of human origin (i.e., such library being based on antibodiestaken from a human natural source).

A “humanized antibody” or functional humanized antibody fragment isdefined herein as one that is (i) derived from a non-human source (e.g.,a transgenic mouse which bears a heterologous immune system), whichantibody is based on a human germline sequence; or (ii) chimeric,wherein the variable domain is derived from a non-human origin and theconstant domain is derived from a human origin or (iii) CDR-grafted,wherein the CDRs of the variable domain are from a non-human origin,while one or more frameworks of the variable domain are of human originand the constant domain (if any) is of human origin.

As used herein, an antibody “binds specifically to,” is “specificto/for” or “specifically recognizes” an antigen (here, CD38) if suchantibody is able to discriminate between such antigen and one or morereference antigen(s), since binding specificity is not an absolute, buta relative property. In its most general form (and when no definedreference is mentioned), “specific binding” is referring to the abilityof the antibody to discriminate between the antigen of interest and anunrelated antigen, as determined, for example, in accordance with one ofthe following methods. Such methods comprise, but are not limited toWestern blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. Forexample, a standard ELISA assay can be carried out. The scoring may becarried out by standard color development (e.g. secondary antibody withhorseradish peroxide and tetramethyl benzidine with hydrogenperoxide).The reaction in certain wells is scored by the optical density, forexample, at 450 nm. Typical background (=negative reaction) may be 0.1OD; typical positive reaction may be 1 OD. This means the differencepositive/negative can be more than 10-fold. Typically, determination ofbinding specificity is performed by using not a single referenceantigen, but a set of about three to five unrelated antigens, such asmilk powder, BSA, transferrin or the like.

However, “specific binding” also may refer to the ability of an antibodyto discriminate between the target antigen and one or more closelyrelated antigen(s), which are used as reference points, e.g. betweenCD38 and CD157. Additionally, “specific binding” may relate to theability of an antibody to discriminate between different parts of itstarget antigen, e.g. different domains or regions of CD38, such asepitopes in the N-terminal or in the C-terminal region of CD38, orbetween one or more key amino acid residues or stretches of amino acidresidues of CD38.

Also, as used herein, an “immunoglobulin” (Ig) hereby is defined as aprotein belonging to the class IgG, IgM, IgE, IgA, or IgD (or anysubclass thereof), and includes all conventionally known antibodies andfunctional fragments thereof. A “functional fragment” of anantibody/immunoglobulin hereby is defined as a fragment of anantibody/immunoglobulin (e.g., a variable region of an IgG) that retainsthe antigen-binding region. An “antigen-binding region” of an antibodytypically is found in one or more hypervariable region(s) of anantibody, i.e., the CDR-1, -2, and/or -3 regions; however, the variable“framework” regions can also play an important role in antigen binding,such as by providing a scaffold for the CDRs. Preferably, the“antigen-binding region” comprises at least amino acid residues 4 to 103of the variable light (VL) chain and 5 to 109 of the variable heavy (VH)chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111of VH, and particularly preferred are the complete VL and VH chains(amino acid positions 1 to 109 of VL and 1 to 113 of VH; numberingaccording to WO 97/08320). A preferred class of immunoglobulins for usein the present invention is IgG. “Functional fragments” of the inventioninclude the domain of a F(ab′)2 fragment, a Fab fragment and scFv. TheF(ab′)2 or Fab may be engineered to minimize or completely remove theintermolecular disulphide interactions that occur between the Cm and CLdomains.

An antibody of the invention may be derived from a recombinant antibodylibrary that is based on amino acid sequences that have been designed insilico and encoded by nucleic acids that are synthetically created. Insilico design of an antibody sequence is achieved, for example, byanalyzing a database of human sequences and devising a polypeptidesequence utilizing the data obtained therefrom. Methods for designingand obtaining in silico-created sequences are described, for example, inKnappik et al., J. Mol. Biol. (2000) 296:57; Krebs et al., J. Immunol.Methods. (2001) 254:67; and U.S. Pat. No. 6,300,064 issued to Knappik etal., which hereby are incorporated by reference in their entirety.

Antibodies of the Invention

Throughout this document, reference is made to the followingrepresentative antibodies of the invention: “antibody nos.” or “LACS” or“MOR” 3077, 3079, 3080 and 3100. LAC 3077 represents an antibody havinga variable heavy region corresponding to SEQ ID NO: 1 (DNA)/SEQ ID NO: 5(protein) and a variable light region corresponding to SEQ ID NO: 9(DNA)/SEQ ID NO: 13 (protein). LAC 3079 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 2 (DNA)/SEQ ID NO: 6(protein) and a variable light region corresponding to SEQ ID NO: 10(DNA)/SEQ ID NO: 14 (protein). LAC 3080 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 3 (DNA)/SEQ ID NO: 7(protein) and a variable light region corresponding to SEQ ID NO: 11(DNA)/SEQ ID NO: 15 (protein). LAC 3100 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 4 (DNA)/SEQ ID NO: 8(protein) and a variable light region corresponding to SEQ ID NO: 12(DNA)/SEQ ID NO: 16 (protein).

In one aspect, the invention provides antibodies having anantigen-binding region that can bind specifically to or has a highaffinity for one or more regions of CD38, whose amino acid sequence isdepicted by SEQ ID NO: 22. An antibody is said to have a “high affinity”for an antigen if the affinity measurement is at least 100 nM(monovalent affinity of Fab fragment). An inventive antibody orantigen-binding region preferably can bind to CD38 with an affinity ofabout less than 100 nM, more preferably less than about 60 nM, and stillmore preferably less than about 30 nM. Further preferred are antibodiesthat bind to CD38 with an affinity of less than about 10 nM, and morepreferably less than 3 about nM. For instance, the affinity of anantibody of the invention against CD38 may be about 10.0 nM or 2.4 nM(monovalent affinity of Fab fragment).

Table 1 provides a summary of affinities of representative antibodies ofthe invention, as determined by surface plasmon resonance (Biacore) andFACS Scatchard analysis:

TABLE 1 Antibody Affinities BIACORE FACS Scatchard Antibody (Fab)(IgG1)^(b) (Fab or IgG1) K_(D) [nM]^(a) K_(D) [nM]^(a) MOR03077 56.00.89 MOR03079 2.4 0.60 MOR03080 27.5 0.47 MOR03100 10.0 6.31 ChimericOKT10 not determined 8.28 ^(a)mean from at least 2 different affinitydeterminations ^(b)RPMI8226 MM cell-line used for FACS-Scatchards

With reference to Table 1, the affinity of LACs 3077, 3079, 3080 and3100 was measured by surface plasmon resonance (Biacore) on immobilizedrecombinant CD38 and by a flow cytometry procedure utilizing theCD38-expressing human RPMI8226 cell line. The Biacore studies wereperformed on directly immobilized antigen (CD38-Fc fusion protein). TheFab format of LACs 3077, 3079, 3080 and 3100 exhibit an monovalentaffinity range between about 2.4 and 56 nM on immobilized CD38-Fc fusionprotein with LAC 3079 showing the highest affinity, followed by Fabs3100, 3080 and 3077.

The IgG1 format was used for the cell-based affinity determination (FACSScatchard). The right column of Table 1 denotes the binding strength ofthe LACS in this format. LAC 3080 showed the strongest binding, which isslightly stronger than LACS 3079 and 3077.

Another preferred feature of preferred antibodies of the invention istheir specificity for an area within the N-terminal region of CD38. Forexample, LACs 3077, 3079, 3080, and 3100 of the invention can bindspecifically to the N-terminal region of CD38.

The type of epitope to which an antibody of the invention binds may belinear (i.e. one consecutive stretch of amino acids) or conformational(i.e. multiple stretches of amino acids). In order to determine whetherthe epitope of a particular antibody is linear or conformational, theskilled worker can analyze the binding of antibodies to overlappingpeptides (e.g., 13-mer peptides with an overlap of 11 amino acids)covering different domains of CD38. Using this analysis, the inventorshave discovered that LACS 3077, 3080, and 3100 recognize discontinuousepitopes in the N-terminal region of CD38, whereas the epitope of LAC3079 can be described as linear (see FIG. 7). Combined with theknowledge provided herein, the skilled worker in the art will know howto use one or more isolated epitopes of CD38 for generating antibodieshaving an antigen-binding region that is specific for said epitopes(e.g. using synthetic peptides of epitopes of CD38 or cells expressingepitopes of CD38).

An antibody of the invention preferably is species cross-reactive withhumans and at least one other species, which may be a rodent species ora non-human primate. The non-human primate can be rhesus, baboon and/orcynomolgus. The rodent species can be mouse, rat and/or hamster. Anantibody that is cross reactive with at least one rodent species, forexample, can provide greater flexibility and benefits over knownanti-CD38 antibodies, for purposes of conducting in vivo studies inmultiple species with the same antibody.

Preferably, an antibody of the invention not only is able to bind toCD38, but also is able to mediate killing of a cell expressing CD38.More specifically, an antibody of the invention can mediate itstherapeutic effect by depleting CD38-positive (e.g., malignant) cellsvia antibody-effector functions. These functions includeantibody-dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity (CDC).

Table 2 provides a summary of the determination of EC50 values ofrepresentative antibodies of the invention in both ADCC and CDC:

TABLE 2 EC50 Values of Antibodies ADCC CDC Antibody EC50 [nM] EC50 [nM](IgG1) LP-1 RPMI8226 CHO-transfectants MOR03077 0.60a 0.08a 0.8c; 0.94dMOR03079 0.09^(a) 0.04^(a) 0.41^(c) MOR03080 0.17^(b) 0.05^(a) 3.2^(c);2.93^(d) MOR03100 1.00^(b) 0.28^(a) 10.9^(c); 13.61^(e) Chimeric5.23^(a) 4.10^(a) 9.30^(c) OKT10 ^(a)mean from at least 2 EC50determinations ^(b)single determination ^(c)mean from 2 EC50determinations ^(d)mean from 3 EC50 determinations ^(e)mean from 4 EC50determinations

CD38-expression, however, is not only found on immune cells within themyeloid (e.g. monocytes, granulocytes) and lymphoid lineage (e.g.activated B and T-cells; plasma cells), but also on the respectiveprecursor cells. Since it is important that those cells are not affectedby antibody-mediated killing of malignant cells, the antibodies of thepresent invention are preferably not cytotoxic to precursor cells.

In addition to its catalytic activities as a cyclic ADP-ribose cyclaseand hydrolase, CD38 displays the ability to transduce signals ofbiological relevance (Hoshino et al., 1997; Ausiello et al., 2000).Those functions can be induced in vivo by, e.g. receptor-ligandinteractions or by cross-linking with agonistic anti-CD38 antibodies,leading, e.g. to calcium mobilization, lymphocyte proliferation andrelease of cytokines. Preferably, the antibodies of the presentinvention are non-agonistic antibodies.

Peptide Variants

Antibodies of the invention are not limited to the specific peptidesequences provided herein. Rather, the invention also embodies variantsof these polypeptides. With reference to the instant disclosure andconventionally available technologies and references, the skilled workerwill be able to prepare, test and utilize functional variants of theantibodies disclosed herein, while appreciating that variants having theability to mediate killing of a CD38+ target cell fall within the scopeof the present invention. As used in this context, “ability to mediatekilling of a CD38+ target cell” means a functional characteristicascribed to an anti-CD38 antibody of the invention. Ability to mediatekilling of a CD38+ target cell, thus, includes the ability to mediatekilling of a CD38+ target cell, e.g. by ADCC and/or CDC, or by toxinconstructs conjugated to an antibody of the invention.

A variant can include, for example, an antibody that has at least onealtered complementarity determining region (CDR) (hyper-variable) and/orframework (FR) (variable) domain/position, vis-à-vis a peptide sequencedisclosed herein. To better illustrate this concept, a brief descriptionof antibody structure follows.

An antibody is composed of two peptide chains, each containing one(light chain) or three (heavy chain) constant domains and a variableregion (VL, VH), the latter of which is in each case made up of four FRregions and three interspaced CDRs. The antigen-binding site is formedby one or more CDRs, yet the FR regions provide the structural frameworkfor the CDRs and, hence, play an important role in antigen binding. Byaltering one or more amino acid residues in a CDR or FR region, theskilled worker routinely can generate mutated or diversified antibodysequences, which can be screened against the antigen, for new orimproved properties, for example.

Tables 3a (VH) and 3b (VL) delineate the CDR and FR regions for certainantibodies of the invention and compare amino acids at a given positionto each other and to corresponding consensus or “master gene” sequences(as described in U.S. Pat. No. 6,300,064):

TABLE 3a VH Sequences

TABLE 3b VL Sequences

The skilled worker can use the data in Tables 3a and 3b to designpeptide variants that are within the scope of the present invention. Itis preferred that variants are constructed by changing amino acidswithin one or more CDR regions; a variant might also have one or morealtered framework regions. With reference to a comparison of the novelantibodies to each other, candidate residues that can be changed includee.g. residues 4 or 37 of the variable light and e.g. residues 13 or 43of the variable heavy chains of LACs 3080 and 3077, since these arepositions of variance vis-à-vis each other. Alterations also may be madein the framework regions. For example, a peptide FR domain might bealtered where there is a deviation in a residue compared to a germlinesequence.

With reference to a comparison of the novel antibodies to thecorresponding consensus or “master gene” sequence, candidate residuesthat can be changed include e.g. residues 27, 50 or 90 of the variablelight chain of LAC 3080 compared to VLλ3 and e.g. residues 33, 52 and 97of the variable heavy chain of LAC 3080 compared to VH3. Alternatively,the skilled worker could make the same analysis by comparing the aminoacid sequences disclosed herein to known sequences of the same class ofsuch antibodies, using, for example, the procedure described by Knappiket al., 2000 and U.S. Pat. No. 6,300,064 issued to Knappik et al.

Furthermore, variants may be obtained by using one LAC as starting pointfor optimization by diversifying one or more amino acid residues in theLAC, preferably amino acid residues in one or more CDRs, and byscreening the resulting collection of antibody variants for variantswith improved properties. Particularly preferred is diversification ofone or more amino acid residues in CDR-3 of VL, CDR-3 of VH, CDR-1 of VLand/or CDR-2 of VH. Diversification can be done by synthesizing acollection of DNA molecules using trinucleotide mutagenesis (TRIM)technology (Virnekäs, B., Ge, L., Plückthun, A., Schneider, K. C.,Wellnhofer, G., and Moroney S. E. (1994) Trinucleotide phosphoramidites:ideal reagents for the synthesis of mixed oligonucleotides for randommutagenesis. Nucl. Acids Res. 22, 5600.).

Conservative Amino Acid Variants

Polypeptide variants may be made that conserve the overall molecularstructure of an antibody peptide sequence described herein. Given theproperties of the individual amino acids, some rational substitutionswill be recognized by the skilled worker. Amino acid substitutions,i.e., “conservative substitutions,” may be made, for instance, on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.

For example, (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt α-helices. Similarly, certainamino acids, such as alanine, cysteine, leucine, methionine, glutamicacid, glutamine, histidine and lysine are more commonly found inα-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophanand threonine are more commonly found in β-pleated sheets. Glycine,serine, aspartic acid, asparagine, and proline are commonly found inturns. Some preferred substitutions may be made among the followinggroups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given theknown genetic code, and recombinant and synthetic DNA techniques, theskilled scientist readily can construct DNAs encoding the conservativeamino acid variants. In one particular example, amino acid position 3 inSEQ ID NOS: 5, 6, 7, and/or 8 can be changed from a Q to an E.

As used herein, “sequence identity” between two polypeptide sequencesindicates the percentage of amino acids that are identical between thesequences. “Sequence similarity” indicates the percentage of amino acidsthat either are identical or that represent conservative amino acidsubstitutions. Preferred polypeptide sequences of the invention have asequence identity in the CDR regions of at least 60%, more preferably,at least 70% or 80%, still more preferably at least 90% and mostpreferably at least 95%. Preferred antibodies also have a sequencesimilarity in the CDR regions of at least 80%, more preferably 90% andmost preferably 95%.

DNA Molecules of the Invention

The present invention also relates to the DNA molecules that encode anantibody of the invention. These sequences include, but are not limitedto, those DNA molecules set forth in FIGS. 1a and 2 a.

DNA molecules of the invention are not limited to the sequencesdisclosed herein, but also include variants thereof. DNA variants withinthe invention may be described by reference to their physical propertiesin hybridization. The skilled worker will recognize that DNA can be usedto identify its complement and, since DNA is double stranded, itsequivalent or homolog, using nucleic acid hybridization techniques. Italso will be recognized that hybridization can occur with less than 100%complementarity. However, given appropriate choice of conditions,hybridization techniques can be used to differentiate among DNAsequences based on their structural relatedness to a particular probe.For guidance regarding such conditions see, Sambrook et al., 1989(Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning:A laboratory manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, USA) and Ausubel et al., 1995 (Ausubel, F. M., Brent, R.,Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K.eds. (1995). Current Protocols in Molecular Biology. New York: JohnWiley and Sons).

Structural similarity between two polynucleotide sequences can beexpressed as a function of “stringency” of the conditions under whichthe two sequences will hybridize with one another. As used herein, theterm “stringency” refers to the extent that the conditions disfavorhybridization. Stringent conditions strongly disfavor hybridization, andonly the most structurally related molecules will hybridize to oneanother under such conditions. Conversely, non-stringent conditionsfavor hybridization of molecules displaying a lesser degree ofstructural relatedness. Hybridization stringency, therefore, directlycorrelates with the structural relationships of two nucleic acidsequences. The following relationships are useful in correlatinghybridization and relatedness (where T. is the melting temperature of anucleic acid duplex):

-   -   a. T_(m)=69.3+0.41(G+C)%    -   b. The T_(m) of a duplex DNA decreases by 1° C. with every        increase of 1% in the number of mismatched base pairs.    -   c. (T_(m))_(μ2)−(T_(m))_(μ1)=18.5 log₁₀ μ2/μ1        -   where μ1 and μ2 are the ionic strengths of two solutions.

Hybridization stringency is a function of many factors, includingoverall DNA concentration, ionic strength, temperature, probe size andthe presence of agents which disrupt hydrogen bonding. Factors promotinghybridization include high DNA concentrations, high ionic strengths, lowtemperatures, longer probe size and the absence of agents that disrupthydrogen bonding. Hybridization typically is performed in two phases:the “binding” phase and the “washing” phase.

First, in the binding phase, the probe is bound to the target underconditions favoring hybridization. Stringency is usually controlled atthis stage by altering the temperature. For high stringency, thetemperature is usually between 65° C. and 70° C., unless short (<20 nt)oligonucleotide probes are used. A representative hybridization solutioncomprises 6×SSC, 0.5% SDS, 5×Denhardt's solution and 100 μg ofnonspecific carrier DNA. See Ausubel et al., section 2.9, supplement 27(1994). Of course, many different, yet functionally equivalent, bufferconditions are known. Where the degree of relatedness is lower, a lowertemperature may be chosen. Low stringency binding temperatures arebetween about 25° C. and 40° C. Medium stringency is between at leastabout 40° C. to less than about 65° C. High stringency is at least about65° C.

Second, the excess probe is removed by washing. It is at this phase thatmore stringent conditions usually are applied. Hence, it is this“washing” stage that is most important in determining relatedness viahybridization. Washing solutions typically contain lower saltconcentrations. One exemplary medium stringency solution contains 2×SSCand 0.1% SDS. A high stringency wash solution contains the equivalent(in ionic strength) of less than about 0.2×SSC, with a preferredstringent solution containing about 0.1×SSC. The temperatures associatedwith various stringencies are the same as discussed above for “binding.”The washing solution also typically is replaced a number of times duringwashing. For example, typical high stringency washing conditionscomprise washing twice for 30 minutes at 55° C. and three times for 15minutes at 60° C.

Accordingly, the present invention includes nucleic acid molecules thathybridize to the molecules of set forth in FIGS. 1a and 2a under highstringency binding and washing conditions, where such nucleic moleculesencode an antibody or functional fragment thereof having properties asdescribed herein. Preferred molecules (from an mRNA perspective) arethose that have at least 75% or 80% (preferably at least 85%, morepreferably at least 90% and most preferably at least 95%) homology orsequence identity with one of the DNA molecules described herein. In oneparticular example of a variant of the invention, nucleic acid position7 in SEQ ID NOS: 1, 2, 3 and/or 4 can be substituted from a C to a G,thereby changing the codon from CAA to GAA.

Functionally Equivalent Variants

Yet another class of DNA variants within the scope of the invention maybe described with reference to the product they encode (see the peptideslisted in FIGS. 1b and 2b ). These functionally equivalent genes arecharacterized by the fact that they encode the same peptide sequencesfound in FIGS. 1b and 2b due to the degeneracy of the genetic code. SEQID NOS: 1 and 31 are an example of functionally equivalent variants, astheir nucleic acid sequences are different, yet they encode the samepolypeptide, i.e. SEQ ID NO: 5.

It is recognized that variants of DNA molecules provided herein can beconstructed in several different ways. For example, they may beconstructed as completely synthetic DNAs. Methods of efficientlysynthesizing oligonucleotides in the range of 20 to about 150nucleotides are widely available. See Ausubel et al., section 2.11,Supplement 21 (1993). Overlapping oligonucleotides may be synthesizedand assembled in a fashion first reported by Khorana et al., J. Mol.Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section 8.2.Synthetic DNAs preferably are designed with convenient restriction sitesengineered at the 5′ and 3′ ends of the gene to facilitate cloning intoan appropriate vector.

As indicated, a method of generating variants is to start with one ofthe DNAs disclosed herein and then to conduct site-directed mutagenesis.See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typicalmethod, a target DNA is cloned into a single-stranded DNA bacteriophagevehicle. Single-stranded DNA is isolated and hybridized with anoligonucleotide containing the desired nucleotide alteration(s). Thecomplementary strand is synthesized and the double stranded phage isintroduced into a host. Some of the resulting progeny will contain thedesired mutant, which can be confirmed using DNA sequencing. Inaddition, various methods are available that increase the probabilitythat the progeny phage will be the desired mutant. These methods arewell known to those in the field and kits are commercially available forgenerating such mutants.

Recombinant DNA Constructs and Expression

The present invention further provides recombinant DNA constructscomprising one or more of the nucleotide sequences of the presentinvention. The recombinant constructs of the present invention are usedin connection with a vector, such as a plasmid or viral vector, intowhich a DNA molecule encoding an antibody of the invention is inserted.

The encoded gene may be produced by techniques described in Sambrook etal., 1989, and Ausubel et al., 1989. Alternatively, the DNA sequencesmay be chemically synthesized using, for example, synthesizers. See, forexample, the techniques described in OLIGONUCLEOTIDE SYNTHESIS (1984,Gait, ed., IRL Press, Oxford), which is incorporated by reference hereinin its entirety. Recombinant constructs of the invention are comprisedwith expression vectors that are capable of expressing the RNA and/orprotein products of the encoded DNA(s). The vector may further compriseregulatory sequences, including a promoter operably linked to the openreading frame (ORF). The vector may further comprise a selectable markersequence. Specific initiation and bacterial secretory signals also maybe required for efficient translation of inserted target gene codingsequences.

The present invention further provides host cells containing at leastone of the DNAs of the present invention. The host cell can be virtuallyany cell for which expression vectors are available. It may be, forexample, a higher eukaryotic host cell, such as a mammalian cell, alower eukaryotic host cell, such as a yeast cell, but preferably is aprokaryotic cell, such as a bacterial cell. Introduction of therecombinant construct into the host cell can be effected by calciumphosphate transfection, DEAE, dextran mediated transfection,electroporation or phage infection.

Bacterial Expression

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and, if desirable, to provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus.

Bacterial vectors may be, for example, bacteriophage-, plasmid- orphagemid-based. These vectors can contain a selectable marker andbacterial origin of replication derived from commercially availableplasmids typically containing elements of the well known cloning vectorpBR322 (ATCC 37017). Following transformation of a suitable host strainand growth of the host strain to an appropriate cell density, theselected promoter is de-repressed/induced by appropriate means (e.g.,temperature shift or chemical induction) and cells are cultured for anadditional period. Cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the proteinbeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of antibodies or to screen peptidelibraries, for example, vectors which direct the expression of highlevels of fusion protein products that are readily purified may bedesirable.

Therapeutic Methods

Therapeutic methods involve administering to a subject in need oftreatment a therapeutically effective amount of an antibody contemplatedby the invention. A “therapeutically effective” amount hereby is definedas the amount of an antibody that is of sufficient quantity to depleteCD38-positive cells in a treated area of a subject—either as a singledose or according to a multiple dose regimen, alone or in combinationwith other agents, which leads to the alleviation of an adversecondition, yet which amount is toxicologically tolerable. The subjectmay be a human or non-human animal (e.g., rabbit, rat, mouse, monkey orother lower-order primate).

An antibody of the invention might be co-administered with knownmedicaments, and in some instances the antibody might itself bemodified. For example, an antibody could be conjugated to an immunotoxinor radioisotope to potentially further increase efficacy.

The inventive antibodies can be used as a therapeutic or a diagnostictool in a variety of situations where CD38 is undesirably expressed orfound. Disorders and conditions particularly suitable for treatment withan antibody of the inventions are multiple myeloma (MM) and otherhaematological diseases, such as chronic lymphocytic leukemia (CLL),chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML),and acute lymphocytic leukemia (ALL). An antibody of the invention alsomight be used to treat inflammatory disease such as rheumatoid arthritis(RA) or systemic lupus erythematosus (SLE).

To treat any of the foregoing disorders, pharmaceutical compositions foruse in accordance with the present invention may be formulated in aconventional manner using one or more physiologically acceptablecarriers or excipients. An antibody of the invention can be administeredby any suitable means, which can vary, depending on the type of disorderbeing treated. Possible administration routes include parenteral (e.g.,intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous), intrapulmonary and intranasal, and, if desired for localimmunosuppressive treatment, intralesional administration. In addition,an antibody of the invention might be administered by pulse infusion,with, e.g., declining doses of the antibody. Preferably, the dosing isgiven by injections, most preferably intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic. The amount to be administered will depend on a variety offactors such as the clinical symptoms, weight of the individual, whetherother drugs are administered. The skilled artisan will recognize thatthe route of administration will vary depending on the disorder orcondition to be treated.

Determining a therapeutically effective amount of the novel polypeptide,according to this invention, largely will depend on particular patientcharacteristics, route of administration, and the nature of the disorderbeing treated. General guidance can be found, for example, in thepublications of the International Conference on Harmonisation and inREMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528(18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990).More specifically, determining a therapeutically effective amount willdepend on such factors as toxicity and efficacy of the medicament.Toxicity may be determined using methods well known in the art and foundin the foregoing references. Efficacy may be determined utilizing thesame guidance in conjunction with the methods described below in theExamples.

Diagnostic Methods

CD38 is highly expressed on hematological cells in certain malignancies;thus, an anti-CD38 antibody of the invention may be employed in order toimage or visualize a site of possible accumulation of malignant cells ina patient. In this regard, an antibody can be detectably labeled,through the use of radioisotopes, affinity labels (such as biotin,avidin, etc.) fluorescent labels, paramagnetic atoms, etc. Proceduresfor accomplishing such labeling are well known to the art. Clinicalapplication of antibodies in diagnostic imaging are reviewed byGrossman, H. B., Urol. Clin. North Amer. 13:465-474 (1986)), Unger, E.C. et al., Invest. Radiol. 20:693-700 (1985)), and Khaw, B. A. et al.,Science 209:295-297 (1980)).

The detection of foci of such detectably labeled antibodies might beindicative of a site of tumor development, for example. In oneembodiment, this examination is done by removing samples of tissue orblood and incubating such samples in the presence of the detectablylabeled antibodies. In a preferred embodiment, this technique is done ina non-invasive manner through the use of magnetic imaging, fluorography,etc. Such a diagnostic test may be employed in monitoring the success oftreatment of diseases, where presence or absence of CD38-positive cellsis a relevant indicator. The invention also contemplates the use of ananti-CD38 antibody, as described herein for diagnostics in an ex vivosetting.

Therapeutic and Diagnostic Compositions

The antibodies of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, whereinan antibody of the invention (including any functional fragment thereof)is combined in a mixture with a pharmaceutically acceptable carriervehicle. Suitable vehicles and their formulation are described, forexample, in REMINGTON'S PHARMACEUTICAL SCIENCES (18th ed., Alfonso R.Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). In order to form apharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofone or more of the antibodies of the present invention, together with asuitable amount of carrier vehicle.

Preparations may be suitably formulated to give controlled-release ofthe active compound. Controlled-release preparations may be achievedthrough the use of polymers to complex or absorb anti-CD38 antibody. Thecontrolled delivery may be exercised by selecting appropriatemacromolecules (for example polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinyl-acetate, methylcellulose,carboxymethylcellulose, or protamine, sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled release preparations is to incorporate anti-CD38antibody into particles of a polymeric material such as polyesters,polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetatecopolymers. Alternatively, instead of incorporating these agents intopolymeric particles, it is possible to entrap these materials inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatine-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences (1980).

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules, orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compositions may, if desired, be presented in a pack or dispenserdevice, which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The invention further is understood by reference to the followingworking examples, which are intended to illustrate and, hence, not limitthe invention.

EXAMPLES Cell-Lines

The following cell-lines were obtained from the European Collection ofCell Cultures (ECACC), the German Collection of Microorganisms (DSMZ) orthe American Type Culture collection (ATCC): hybridoma cell lineproducing the CD38 mouse IgG1 monoclonal antibody OKT10 (ECACC,#87021903), Jurkat cells (DSMZ, ACC282), LP-1 (DSMZ, ACC41), RPMI8226(ATCC, CCL-155), HEK293 (ATCC, CRL-1573), CHO-K1 (ATCC, CRL-61) and Raji(ATCC, CCL-86)

Cells and Culture-Conditions

All cells were cultured under standardized conditions at 37° C. and 5%CO₂ in a humidified incubator. The cell-lines LP-1, RPMI8226, Jurkat andRaji were cultured in RPMI1640 (Pan biotech GmbH, #P04-16500)supplemented with 10% FCS (PAN biotech GmbH, #P30-3302), 50 U/mlpenicillin, 50 μg/ml streptomycin (Gibco, #15140-122) and 2 mM glutamine(Gibco, #25030-024) and, in case of Jurkat- and Raji-cells, additionally10 mM Hepes (Pan biotech GmbH, #P05-01100) and 1 mM sodium pyruvate (Panbiotech GmbH, # P04-43100) had to be added.

CHO-K1 and HEK293 were grown in DMEM (Gibco, #10938-025) supplementedwith 2 mM glutamine and 10% FCS. Stable CD38 CHO-K1 transfectants weremaintained in the presence of G418 (PAA GmbH, P11-012) whereas forHEK293 the addition of 1 mM sodium-pyruvate was essential. Aftertransient transfection of HEK293 the 10% FCS was replaced by Ultra lowIgG FCS (Invitrogen, #16250-078). The cell-line OKT10 was cultured inIDMEM (Gibco, #31980-022), supplemented with 2 mM glutamine and 20% FCS.

Preparation of Single Cell Suspensions from Peripheral Blood

All blood samples were taken after informed consent. Peripheral bloodmononuclear cells (PBMC) were isolated by Histopaque®-1077 (Sigma)according to the manufacturer's instructions from healthy donors. Redblood cells were depleted from these cell suspensions by incubation inACK Lysis Buffer (0.15 M NH4Cl, 10 mM KHCO₃, 0.1 M EDTA) for 5 min at RTor a commercial derivative (Bioscience, #00-4333). Cells were washedtwice with PBS and then further processed for flow cytometry or ADCC(see below).

Flow Cytometry (“FACS”)

All stainings were performed in round bottom 96-well culture plates(Nalge Nunc) with 2×10⁵ cells per well. Cells were incubated with Fab orIgG antibodies at the indicated concentrations in 50 μl FACS buffer(PBS, 3% FCS, 0.02% NaN₃) for 40 min at 4° C. Cells were washed twiceand then incubated with R-Phycoerythrin (PE) conjugated goat-anti-humanor goat-anti-mouse IgG (H+L) F(ab′)₂ (Jackson Immuno Research), diluted1:200 in FACS buffer, for 30 min at 4° C. Cells were again washed,resuspended in 0.3 ml FACS buffer and then analyzed by flow cytometry ina FACSCalibur (Becton Dickinson, San Diego, Calif.).

For FACS based Scatchard analyses RPMI8226 cells were stained with at 12different dilutions (1:2^(n)) starting at 12.5 μg/ml (IgG) finalconcentration. At least two independent measurements were used for eachconcentration and K_(D) values extrapolated from median fluorescenceintensities according to Chamow et al. (1994).

Surface Plasmon Resonance

The kinetic constants k_(on) and k_(off) were determined with serialdilutions of the respective Fab binding to covalently immobilizedCD38-Fc fusion protein using the BIACORE 3000 instrument (BIACORE,Uppsala, Sweden). For covalent antigen immobilization standard EDC-NHSamine coupling chemistry was used. For direct coupling of CD38 Fc-fusionprotein CM5 senor chips (BIACORE) were coated with −600-700 RU in 10 mMacetate buffer, pH 4.5.

For the reference flow cell a respective amount of HSA (human serumalbumin) was used. Kinetic measurements were done in PBS (136 mM NaCl,2.7 mM KCl, 10 mM Na₂HPO4, 1.76 mM KH₂PO₄ pH 7.4) at a flow rate of 20μl/min using Fab concentration range from 1.5-500 nM. Injection time foreach concentration was 1 min, followed by 2 min dissociation phase. Forregeneration 5 μl 10 mM HCl was used. All sensograms were fitted locallyusing BIA evaluation software 3.1 (BIACORE).

Example 1 Antibody Generation from HuCAL® Libraries

For the generation of therapeutic antibodies against CD38, selectionswith the MorphoSys HUCAL GOLD® phage display library were carried out.HUCAL GOLD® is a Fab library based on the HUCAL® concept (Knappik etal., 2000; Krebs et al., 2001), in which all six CDRs are diversified,and which employs the CYSDISPLAY™ technology for linking Fab fragmentsto the phage surface (Lohning, 2001).

A. Phagemid Rescue, Phage Amplification and Purification

HUCAL GOLD® phagemid library was amplified in 2×TY medium containing 34μg/ml chloramphenicol and 1% glucose (2×TY-CG). After helper phageinfection (VCSM13) at an OD600 of 0.5 (30 min at 37° C. without shaking;30 min at 37° C. shaking at 250 rpm), cells were spun down (4120 g; 5min; 4° C.), resuspended in 2×TY/34 μg/ml chloramphenicol/50[tg/mlkanamycin and grown overnight at 22° C. Phages werePEG-precipitated from the supernatant, resuspended in PBS/20% glyceroland stored at −80° C. Phage amplification between two panning rounds wasconducted as follows: mid-log phase TGI cells were infected with elutedphages and plated onto LB-agar supplemented with 1% of glucose and 34μg/ml of chloramphenicol (LB-CG). After overnight incubation at 30° C.,colonies were scraped off, adjusted to an OD600 of 0.5 and helper phageadded as described above.

B. Pannings with HUCAL GOLD®

For the selections HUCAL GOLD® antibody-phages were divided into threepools corresponding to different VH master genes VH1/5λκ, pool 2: VH3λκ, pool 3: VH2/4/6λκ). These pools were individually subjected to 3rounds of whole cell panning on CD38-expressing CHO-K1 cells followed bypH-elution and a post-adsorption step on CD38-negative CHO-K1-cells fordepletion of irrelevant antibody-phages. Finally, the remaining antibodyphages were used to infect E. coli TGI cells. After centrifugation thebacterial pellet was resuspended in 2×TY medium, plated on agar platesand incubated overnight at 30° C. The selected clones were then scrapedfrom the plates, phages were rescued and amplified. The second and thethird round of selections were performed as the initial one.

The Fab encoding inserts of the selected HUCAL GOLD® phages weresubcloned into the expression vector pMORPH0×9 Fab FS (Rauchenberger etal., 2003) to facilitate rapid expression of soluble Fab. The DNA of theselected clones was digested with Xbal and EcoRI thereby cutting out theFab encoding insert (ompA-VLCL and phoA-Fd), and cloned into theXbal/EcoRI cut vector pMORPH0×9 Fab FS. Fab expressed in this vectorcarry two C-terminal tags (FLAG™ and STREP-TAG® II) for detection andpurification.

Example 2 Biological Assays

Antibody dependent cellular cytotoxicity (ADCC) and complement-dependentcytotoxicity was measured according to a published protocol based onflow-cytometry analysis (Naundorf et al., 2002) as follows:

ADCC:

For ADCC measurements, target cells (T) were adjusted to 2.0E±05cells/ml and labeled with 100 ng/ml Calcein AM (Molecular Probes,C-3099) in RPMI1640 medium (Pan biotech GmbH) for 2 minutes at roomtemperature. Residual calcein was removed by 3 washing steps in RPMI1640medium. In parallel PBMC were prepared as source for (natural killer)effector cells (E), adjusted to 1.0E+07 and mixed with the labeledtarget cells to yield a final E:T-ratio of 50:1 or less, depending onthe assay conditions. Cells were washed once and the cell-mixresuspended in 200 μl RPMI1640 medium containing the respective antibodyat different dilutions. The plate was incubated for 4 hrs understandardized conditions at 37° C. and 5% CO₂ in a humidified incubator.Prior to FACS analysis cells were labeled with propidium-iodide (PI) andanalyzed by flow-cytometry (Becton-Dickinson). Between 50.000 and150.000 events were counted for each assay.

The following equation gave rise to the killing activity [in %]:

$\frac{{ED}^{A}}{{EL}^{A} + {ED}^{A}} \times 100$

with ED^(A)=events dead cells (calcein+PI stained cells), andEL^(A)=events living cells (calcein stained cells)

CDC:

For CDC measurements, 5.0E+04 CD38 CHO-K1 transfectants were added to amicrotiter well plate (Nunc) together with a 1:4 dilution of human serum(Sigma, #S-1764) and the respective antibody. All reagents and cellswere diluted in RPMI1640 medium (Pan biotech GmbH) supplemented with 10%FCS. The reaction-mix was incubated for 2 hrs under standardizedconditions at 37° C. and 5% CO₂ in a humidified incubator. As negativecontrols served either heat-inactivated complement or CD38-transfectantswithout antibody. Cells were labeled with PI and subjected toFACS-analysis.

In total 5000 events were counted and the number of dead cells atdifferent antibody concentrations used for the determination of EC50values. The following equation gave rise to the killing activity [in %]:

$\frac{{ED}^{C}}{{EL}^{C} + {ED}^{C}} \times 100$

with ED^(C)=events dead cells (PI stained cells), andEL^(C)=events living cells (unstained)

Cytotoxicity values from a total of 12 different antibody-dilutions(1:2^(n)) in triplicates were used in ADCC and duplicates in CDC foreach antibody in order obtain EC-50 values with a standard analysissoftware (PRISM®, Graph Pad Software).

Example 3 Generation of Stable CD38-Transfectants and CD38 Fc-FusionProteins

In order to generate CD38 protein for panning and screening twodifferent expression systems had to be established. The first strategyincluded the generation of CD38-Fc-fusion protein, which was purifiedfrom supernatants after transient transfection of HEK293 cells. Thesecond strategy involved the generation of a stable CHO-K1-cell line forhigh CD38 surface expression to be used for selection of antibody-phagesvia whole cell panning.

As an initial step Jurkat cells (DSMZ ACC282) were used for thegeneration of cDNA (Invitrogen) followed by amplification of the entireCD38-coding sequence using primers complementary to the first 7 and thelast 9 codons of CD38, respectively (primer MTE001 & MTE002rev; Table4). Sequence analysis of the CD38-insert confirmed the published aminoacid sequence by Jackson et al. (1990) except for position 49 whichrevealed a glutamine instead of a tyrosine as described by Nata et al.(1997). For introduction of restriction endonuclease sites and cloninginto different derivatives of expression vector pcDNA3.1 (Stratagene),the purified PCR-product served as a template for the re-amplificationof the entire gene (primers MTE006 & MTE007rev, Table 4) or a part(primers MTE004 & MTE009rev, Table 4) of it. In the latter case afragment encoding for the extracellular domain (aa 45 to 300) wasamplified and cloned in frame between a human Vkappa leader sequence anda human Fc-gamma 1 sequence. This vector served as expression vector forthe generation of soluble CD38-Fc fusion-protein. AnotherpcDNA3.1-derivative without leader-sequence was used for insertion ofthe CD38 full-length gene. In this case a stop codon in front of theFc-coding region and the missing leader-sequence gave rise toCD38-surface expression. HEK293 cells were transiently transfected withthe Fc-fusion protein vector for generation of soluble CD38 Fc-fusionprotein and, in case of the full-length derivative, CHO-K1-cells weretransfected for the generation of a stable CD38-expressing cell line.

TABLE 4  Primer # Sequence (5′->3′) MTE001 ATG GCC AAC TGC GAG TTC AGC(SEQ ID NO: 25) MTE002rev TCA GAT CTC AGA TGT GCA AGA TGA ATC(SEQ ID NO: 26) MTE004 TT GGT ACC AGG TGG CGC CAG CAG TG (SEQ ID NO: 27)MTE006 TT GGT ACC ATG GCC AAC TGC GAG (SEQ ID NO: 28) MTE007revCCG ATA TCA* GAT CTC AGA TGT GCA AGA TG (SEQ ID NO: 29) MTE009revCCG ATA TC   GAT CTC AGA TGT GCA AGA TG (SEQ ID NO: 30) *leading to astop codon (TGA) in the sense orientation.

Example 4 Cloning, Expression and Purification of HuCAL® IgG1

In order to express full length IgG, variable domain fragments of heavy(VH) and light chains (VL) were subcloned from Fab expression vectorsinto appropriate pMORPH®_hIg vectors (see FIGS. 8 to 10). Restrictionendonuclease pairs BlpI/MfeI (insert-preparation) and BlpI/EcoRI(vector-preparation) were used for subcloning of the VH domain fragmentinto pMORPH®_hIgG1. Enzyme-pairs EcoRV/HpaI (lambda-insert) andEcoRV/BsiWI (kappa-insert) were used for subcloning of the VL domainfragment into the respective pMORPH®_hIgκ_1 or pMORPH®_h_Igλ_1 vectors.Resulting IgG constructs were expressed in HEK293 cells (ATCC CRL-1573)by transient transfection using standard calcium phosphate-DNAcoprecipitation technique.

IgGs were purified from cell culture supernatants by affinitychromatography via Protein A Sepharose column. Further down streamprocessing included a buffer exchange by gel filtration and sterilefiltration of purified IgG. Quality control revealed a purity of >90% byreducing SDS-PAGE and >90% monomeric IgG as determined by analyticalsize exclusion chromatography. The endotoxin content of the material wasdetermined by a kinetic LAL based assay (Cambrex European EndotoxinTesting Service, Belgium).

Example 5 Generation and Production of Chimeric OKT10 (chOKT10; SEQ IDNOS: 23 and 24)

For the construction of chOKT10 the mouse VH and VL regions wereamplified by PCR using cDNA prepared from the murine OKT10 hybridomacell line (ECACC #87021903). A set of primers was used as published(Dattamajumdar et al., 1996; Zhou et al., 1994). PCR products were usedfor Topo-cloning (Invitrogen; pCRII-vector) and single coloniessubjected to sequence analysis (M13 reverse primer) which revealed twodifferent kappa light chain sequences and one heavy chain sequence.According to sequence alignments (EMBL-nucleotide sequence database) andliterature (Krebber et al, 1997) one of the kappa-sequence belongs tothe intrinsic repertoire of the tumor cell fusion partner X63Ag8.653 andhence does not belong to OKT10 antibody. Therefore, only the new kappasequence and the single VH-fragment was used for further cloning. Bothfragments were reamplified for the addition of restriction endonucleasesites followed by cloning into the respective pMORPH® IgG1-expressionvectors. The sequences for the heavy chain (SEQ ID NO: 23) and lightchain (SEQ ID NO: 24) are given in FIG. 6. HEK293 cells were transfectedtransiently and the supernatant analyzed in FACS for the chimeric OKT10antibody binding to the CD38 overexpressing Raji cell line (ATCC).

Example 6 Epitope Mapping 1. Materials and Methods: Antibodies:

The following anti-CD38 IgGs were sent for epitope mappings:

Conc. [mg/ml]/ MOR# Lot # Format Vol. [μl] MOR03077 2CHE106_030602 humanIgG1 0.44/1500 MOR03079 2APO31 human IgG1 0.38/500 MOR03080030116_4CUE16 human IgG1 2.28/200 MOR03100 030612_6SBA6 human IgG10.39/500 chim. 030603_2CHE111 human IgG1 0.83/500 OKT10* *chimeric OKT10consisting of human Fc and mouse variable regions.

CD38-Sequence:

The amino acid (aa) sequence (position 44-300) is based on human CD38taken from the published sequence under SWISS-PROT primary accessionnumber P28907. At position 49 the aa Q (instead of T) has been used forthe peptide-design.

PepSpot-Analysis:

The antigen peptides were synthesized on a cellulose membrane in astepwise manner resulting in a defined arrangement (peptide array) andare covalently bound to the cellulose membrane. Binding assays wereperformed directly on the peptide array.

In general an antigen peptide array is incubated with blocking bufferfor several hours to reduce non-specific binding of the antibodies. Theincubation with the primary (antigen peptide-binding) antibody inblocking buffer occurs followed by the incubation with the peroxidase(POD)-labelled secondary antibody, which binds selectively the primaryantibody. A short T (Tween)-TBS-buffer washing directly after theincubation of the antigen peptide array with the secondary antibodyfollowed by the first chemiluminescence experiment is made to get afirst overview which antigen peptides do bind the primary antibody.Several buffer washing steps follow (T-TBS- and TBS-buffer) to reducefalse positive binding (unspecific antibody binding to the cellulosemembrane itself). After these washing steps the final chemiluminescenceanalysis is performed. The data were analysed with an imaging systemshowing the signal intensity (Boehringer Light units, BLU) as singlemeasurements for each peptide. In order to evaluate non-specific bindingof the secondary antibodies (anti-human IgG), these antibodies wereincubated with the peptide array in the absence of primary antibodies asthe first step. If the primary antibody does not show any binding to thepeptides it can be directly labelled with POD, which increases thesensitivity of the system (as performed for MOR3077). In this case aconventional coupling chemistry via free amino-groups is performed.

The antigen was scanned with 13-mer peptides (11 amino acids overlap).This resulted in arrays of 123 peptides. Binding assays were performeddirectly on the array. The peptide-bound antibodies MOR03077, MOR03079,MOR03080, MOR03100 and chimeric OKT10 were detected using aperoxidase-labelled secondary antibody (peroxidase conjugate-goatanti-human IgG, gamma chain specific, affinity isolated antibody;Sigma-Aldrich, A6029). The mappings were performed with achemiluminescence substrate in combination with an imaging system.Additionally, a direct POD-labelling of MOR03077 was performed in orderto increase the sensitivity of the system.

2. Summary and Conclusions:

All five antibodies showed different profiles in the PepSpot analysis. Aschematic summary is given in FIG. 7, which illustrates the different aasequences of CD38 being recognized. The epitope for MOR03079 andchimeric OKT10 can clearly be considered as linear. The epitope forMOR03079 can be postulated within aa 192-206 (VSRRFAEAACDVVHV (SEQ IDNO:38)) of CD38 whereas for chimeric OKT10 a sequence between aa 284 and298 (FLQCVKNPEDSSCTS (SEQ ID NO:39)) is recognized predominantly. Thelatter results confirm the published data for the parental murine OKT10(Hoshino et al., 1997), which postulate its epitope between aa 280-298.Yet, for a more precise epitope definition and determination of keyamino acids (main antigen-antibody interaction sites) a shortening ofpeptides VSRRFAEAACDVVHV (SEQ ID NO: 38) and FLQCVKNPEDSSCTS (SEQ IDNO:39) and an alanine-scan of both should be envisaged.

The epitopes for MOR03080 and MOR03100 can be clearly considered asdiscontinuous since several peptides covering different sites of theprotein sites were recognized. Those peptides comprise aa 82-94 and aa158-170 for MOR03080 and aa 82-94, 142-154, 158-170, 188-200 and 280-296for MOR03100. However, some overlaps between both epitopes can bepostulated since two different sites residing within aa positions 82-94(CQSVWDAFKGAFI (SEQ ID NO:40); peptide #20) and 158-170 (TWCGEFNTSKINY(SEQ ID NO:41); peptide #58) are recognized by both antibodies.

The epitope for MOR03077 can be considered as clearly different from thelatter two and can be described as multisegmented discontinuous epitope.The epitope includes aa 44-66, 110-122, 148-164, 186-200 and 202-224.

Example 7 IL-6-Release/Proliferation Assay 1. Materials and Methods:

Proliferation- and a IL-6 release assays have been performed accordingto Ausiello et al. (2000) with the following modifications: PBMCs fromdifferent healthy donors (after obtaining informed consent) werepurified by density gradient centrifugation using the Histopaque cellseparation system according to the instructions of the supplier (Sigma)and cultured under standard conditions (5% CO2, 37° C.) in RPMI1640medium, supplemented with 10% FCS and glutamine (“complete RPMI1640”).For both assays the following antibodies were used: HuCAL® anti-CD38IgG1s Mabs MOR03077, MOR03079, and MOR03080, an agonistic murine IgG2amonoclonal antibody (IB4; Malavasi et al., 1984), an irrelevant HuCAL®IgG1 antibody, a matched isotype control (murine IgG2a:anti-trinitrophenol, hapten-specific antibody; cat. #: 555571, cloneG155-178; Becton Dickinson) or a medium control. For the IL-6 releaseassay, 1.0 E+06 PBMCs in 0.5 ml complete RPMI1640 medium were incubatedfor 24 hrs in a 15 ml culture tube (Falcon) in the presence of 20 μg/mlantibodies. Cell culture supernatants were harvested and analysed forIL-6 release using the Quantikine kit according to the manufacturer'sprotocol (R&D systems). For the proliferation assay 2.0E+05 PBMCs wereincubated for 3 days in a 96-well flat bottom plate (Nunc) in thepresence of 20 μg/ml antibodies. Each assay was carried out induplicates. After 4 days BrdU was added to each well and cells incubatedfor an additional 24 hrs at 37° C. prior to cell fixation and DNAdenaturation according to the protocol of the supplier (Roche).Incorporation of BrdU was measured via an anti-BrdU peroxidase-coupledantibody in a chemiluminescence-based setting.

2. Summary and Conclusions: Proliferation Assay:

In addition to its catalytic activities as a cyclic ADP-ribose cyclaseand hydrolase, CD38 displays the ability to transduce signals ofbiological relevance (Hoshino et al., 1997; Ausiello et al., 2000).Those functions can be induced in vivo by e.g. receptor-ligandinteractions or by cross-linking with anti-CD38 antibodies. Thosesignalling events lead e.g. to calcium mobilization, lymphocyteproliferation and release of cytokines. However, this signalling is notonly dependent on the antigenic epitope but might also vary from donorto donor (Ausiello et al., 2000). In the view of immunotherapynon-agonistic antibodies are preferable over agonistic antibodies.Therefore, HuCAL® anti-CD38 antibodies (Mabs MOR03077; MOR03079,MOR03080) were further characterized in a proliferation assay andIL-6-(important MM growth-factor) release assay in comparison to thereference antibody chOKT10 and the agonistic anti-CD38 monoclonalantibody IB4.

As demonstrated in FIG. 11 and FIG. 12 the HUCAL® anti-CD38 antibodiesMab#1, 2 and 3 as well as the reference antibody chOKT10 andcorresponding negative controls showed no or only weak induction ofproliferation and no IL-6-release as compared to the agonistic antibodyIB4.

Example 8 Clonogenic Assay 1. Materials and Methods:

PBMCs harbouring autologous CD34+/CD38+ precursor cells were isolatedfrom healthy individuals (after obtaining informed consent) by densitygradient centrifugation using the Histopaque cell separation systemaccording to the instructions of the supplier (Sigma) and incubated withdifferent HUCAL® IgG1 anti-CD38 antibodies (Mabs MOR03077, MOR03079, andMOR03080) and the positive control (PC) chOKTlO at 10 μg/ml. Medium andan irrelevant HUCAL® IgG1 served as background control. Each ADCC-assayconsisted of 4.0E+05 PBMCs which were incubated for 4 hrs at 37° C. inRPMI1640 medium supplemented with 10% FCS. For the clonogenic assay 2.50ml “complete” methylcellulose (CellSystems) was inoculated with 2.5 E+05cells from the ADCC-assay and incubated for colony-development for atleast 14 days in a controlled environment (37° C.; 5% CO2). Colonieswere analyzed by two independent operators and grouped intoBFU-E+CFU-GEMM (erythroid burst forming units andgranulocyte/erythroid/macrophage/megakaryocyte stem cells) and CFU-GM(granulocyte/macrophage stem cells).

2. Summary and Conclusions:

Since CD38-expression is not only found on immune cells within themyeloid (e.g. monocytes, granulocytes) and lymphoid lineage (e.g.activated B and T-cells; plasma cells) but also on the respectiveprecursor cells (CD34+/CD38+), it is important that those cells are notaffected by antibody-mediated killing. Therefore, a clonogenic assay wasapplied in order to analyse those effects on CD34+/CD38+ progenitors.

PBMCs from healthy donors were incubated with HUCAL® anti-CD38antibodies (Mab#1, Mab#2 and Mab#3) or several controls (irrelevantHUCAL® antibody, medium and reference antibody chOKT10 as positivecontrol) according to a standard ADCC-protocol followed by furtherincubation in conditioned methylcellulose for colony-development. Asshown in FIG. 13 no significant reduction of colony-forming units areshown for all HUCAL® anti-CD38 antibodies as compared to an irrelevantantibody or the reference antibody.

Example 9 ADCC Assays with Different Cell-Lines and Primary MultipleMyeloma Cells 1. Materials and Methods:

Isolation and ADCC of MM-patient samples: Bone marrow aspirates wereobtained from multiple myeloma patients (after obtaining informedconsent). Malignant cells were purified via a standard protocol usinganti-CD138 magnetic beads (Milteny Biotec) after density gradientcentrifugation (Sigma). An ADCC-assay was performed as described before.

2. Summary and Conclusions:

Several cell-lines derived from different malignancies were used in ADCCin order to show the cytotoxic effect of the HUCAL® anti-CD38 antibodieson a broader spectrum of cell-lines including different origins and CD38expression-levels. As shown in FIG. 14, all cells were killed in ADCC atconstant antibody concentrations (5 μg/ml) and E:T ratios at 30:1.Cytotoxicity via ADCC was also shown for several multiple myelomasamples from patients. All HUCAL® anti-CD38 antibodies were able toperform a dose-dependent killing of MM-cells and the EC50-values variedbetween 0.006 and 0.249 nM (FIG. 15).

Example 10 Cross-Reactivity Analysis by FACS and Immunohisto-Chemistry(IHC) 1. Materials and Methods:

IHC with tonsils: For IHC HUCAL® anti-CD38 Mabs and an irrelevantnegative control antibody were converted into the bivalent dHLX-format(Pliickthun & Pack, 1997). 5 μm cryo sections from lymph nodes derivedfrom Cynomolgus monkey, Rhesus monkey and humans (retrieved from thearchives of the Institute of Pathology of the University ofGraz/Austria) were cut with a Leica CM3050 cryostat. Sections wereair-dried for 30 minutes to 1 hour and fixed in ice-cold methanol for 10minutes and washed with PBS. For the detection of the dHLX-format amouse anti-His antibody (Dianova) in combination with the Envision Kit(DAKO) was used. For the detection of the anti-CD38 mouse antibodies(e.g. reference mouse monoclonal OKT10) the Envison kit was used only.

FACS-analysis of lymphocytes: EDTA-treated blood samples were obtainedfrom healthy humans (after obtaining informed consent), from Rhesus andCynomolgus monkeys and subjected to density gradient centrifugationusing the Histopaque cell separation system according to theinstructions of the supplier (Sigma). For FACS-analysis cells from theinterphase were incubated with primary antibodies (HUCAL® anti-CD38 andnegative control Mabs as murine IgG2a or Fab-format, the positivecontrol murine antibody OKT10 and a matched isotype control) followed byincubation with anti-M2 Flag FLAG (Sigma; only for Fab-format) and aphycoerythrin (PE)-labeled anti-mouse conjugate (Jackson Research). FACSanalysis was performed on the gated lymphocyte population.

2. Summary and Conclusions:

HUCAL® anti-CD38 were analyzed for inter-species CD38 cross-reactivity.Whereas all anti-CD38 Mabs were able to detect human CD38 on lymphocytesin FACS and IHC, only MOR03080 together with the positive control OKT10showed an additional reactivity with Cynomolgus and Rhesus monkey CD38(see Table 5: Cross-reactivity analysis).

TABLE 5 Lymphocytes (FACS) and lymph-nodes (IHC) from: Cynomolgus RhesusAntibody Human Monkey Monkey Mab#1 ++ − − Mab#2 ++ − − Mab#3 ++ ++ ++ PC++ ++ ++ NC − − − ++: strong positive staining; −: no staining; NC:negative control; PC: positive control (=reference cMAb)

Example 11 Treatment of Human Myeloma Xenografts in Mice (Using theRPMI8226 Cell Line) with MOR03080 1. Establishment of Subcutaneous MouseModel:

A subcutaneous mouse model for the human myeloma-derived tumor cell lineRPMI8226 in female C.B-17-SCID mice was established as follows byAurigon Life Science GmbH (Tutzing, Germany): on day −1, 0, and 1,anti-asialo GM1 polyclonal antibodies (ASGM) (WAKO-Chemicals), whichdeplete the xenoreactive NK-cells in the SCID mice were appliedintravenously in order to deactivate any residual specific immunereactivity in C.B-17-SCID mice. On day 0, either 5×10⁶ or 1×10⁷ RPMI8226tumor cells in 50 μl PBS were inoculated subcutaneously into the rightflank of mice either treated with ASGM (as described above) or untreated(each group consisting of five mice). Tumor development was similar inall 4 inoculated groups with no significant difference being found fortreatment with or without anti-asialo GM1 antibodies or by inoculationof different cell numbers. Tumors appear to be slowly growing with thetendency of stagnation or oscillation in size for some days. Two tumorsoscillated in size during the whole period of investigation, and onetumor even regarded and disappeared totally from a peak volume of 321mm³. A treatment study with this tumor model should include a highnumber of tumor-inoculated animals per group.

2. Treatment with MOR03080:

2.1 Study Objective

This study was performed by Aurigon Life Science GmbH (Tutzing, Germany)to compare the anti-tumor efficacy of intraperitoneally appliedantibodies (HUCAL® anti-CD38) as compared to the vehicle treatment(PBS). The human antibody hMOR03080 (isotype IgG1) was tested indifferent amounts and treatment schedules. In addition the chimericantibody chMOR03080 (isotype IgG2a: a chimeric antibody comprising thevariable regions of MOR03080 and murine constant regions constructed ina similar way as described in Example 5 for chimeric OKT10 (murine VH/VLand human constant regions)) was tested. The RPMI8226 cancer cell linehad been chosen as a model and was inoculated subcutaneously in femaleSCID mice as described above. The endpoints in the study were bodyweight (b.w.), tumor volume and clinical signs.

2.2 Antibodies and Vehicle

The antibodies were provided ready to use to Aurigon at concentrationsof 2.13 mg/ml (MOR03080 hIgG1) and 1.73 mg/ml (MOR03080 chIgG2a, andstored at −80° C. until application. The antibodies were thawed anddiluted with PBS to the respective end concentration. The vehicle (PBS)was provided ready to use to Aurigon and stored at 4° C. untilapplication.

2.3 Animal Specification

Species: mouse

Strain: Fox chase C.B-17-scid (C.B-Igh-1b/IcrTac)

Number and sex: 75 females

Supplier: Taconic M&B, Bomholtvej 10, DK-8680 Ry

Health status: SPF

Weight ordered: appr. 18 g

Acclimatization: 9 days

2.4 Tumor Cell Line

The tumor cells (RPMI8226 cell line) were grown and transported toAurigon Life Science GmbH, where the cells were splitted and grown foranother cycle. Aurigon prepared the cells for injection on the day ofinoculation. The culture medium used for cell propagation was RPMI 1640supplemented with 5% FCS, 2 mM L-Glutamin and PenStrep. The cells showedno unexpected growth rate or behaviour.

For inoculation, tumor cells were suspended in PBS and adjusted to afinal concentration of 1×10⁷ cells/50 μl in PBS. The tumor cellsuspension was mixed thoroughly before being injected.

2.5 Experimental Procedure

On day 0, 1×10⁷ RPMI8226 tumor cells were inoculated subcutaneously intothe right dorsal flank of 75 SCID mice. A first group was built with 15randomly chosen animals (group 5) directly after inoculation. This groupwas treated with 1 mg/kg b.w. hIgG1-MOR03080 every second day betweenday 14 and 36. From all other 60 animals 4 groups were built with tenanimals in each group on day 31 (tumor volume of about 92 mm³). Groups1-4 were built with comparable means tumor sizes and standarddeviations. An additional group of 5 animals (group 6) was chosenshowing relatively small tumor volumes (tumor volume of about 50 mm³)for comparison with pre-treated group 5 (all but three mice showingtumor volumes of less than 10 mm³, one with about 22 mm³, one with about44 mm³ and one with about 119 mm³). Groups 1 to 4 were treated everysecond day from day 32 to day 68 with either PBS (Vehicle; group 1), 1mg/kg b.w. hIgG1-MOR03080 (group 2) or 5 mg/kg b.w.hIgG1-MOR03080 (group3), or with 5 mg/kg b.w. chIgG2a-MOR03080 (group 4). Group 6 did notreceive any treatment (see Table 6). Tumor volumes, body weight andclinical signs were measured two times a week until end of study.

TABLE 6 No. of Type of Treatment dose Appl. volume Group animalsapplication Substance Schedule [mg/kg] [μl/kg] 1 10 i.p. vehicle everysecond day — 10 (PBS) between day 32 and day 68 2 10 i.p. MOR03080 everysecond day 1 10 human IgG1 between day 32 and day 68 3 10 i.p. MOR03080every second day 5 10 human IgG1 between day 32 and day 68 4 10 i.p.MOR03080 every second day 5 10 chimeric between day 32 and IgG2a day 685 15 i.p. MOR03080 every second day 1 10 human IgG1 between day 14 andday 36 6 5 — — — — —

2.6 Results Clinical Observations and Mortality

No specific tumor or substance related clinical findings or mortalitywere observed. In group 3 (hIgG1 5 mg/kg) four animals died during bloodsampling (one on day 3, one on day 34; two on day 52). In group 4(muIgG2a 1 mg/kg) a single animal died during blood sampling (day 34).All other animals, that died during the study have been euthanizedbecause of the tumor size.

Body Weight Development

No drug related interference with weight development was observed incomparison to group 1 (vehicle). Body weight was markedly influenced byblood sampling in groups 3 (hIgG1 5 mg/kg) and 4 (muIgG2a 5 mg/kg).Despite such interruptions the mean weight gain of all groups wascontinuous.

Tumor Development (See FIG. 16)

In group 1 (vehicle) tumor growth was found in the expected rate with aslow progression. As this cell line has a pronounced standard deviationvalues for the largest and smallest tumor have been excluded fromfurther statistical analysis. The tumor growth of animals in group 1 wascomparable to the tumor growth in group 6 (untreated), although thisgroup started with a lower mean tumor volume on day 31. Treatment mighttherefore have a slight influence on the tumor growth rate. In group 1,two mice had to be euthanized before day 83 because of the tumor size,and a further one before day 87, so that the mean value of tumor volumeis no longer representative after day 80. In group 6, one mouse had tobe euthanized before day 80 because of the tumor size, two mice beforeday 83, and a further one before day 87, so that the mean value of tumorvolume is no longer representative after day 76.

In group 2, treated with 1 mg/kg b.w. of hIgG1, one animal has beenexcluded from further analysis, because the tumor grew into the musculartissue and this usually enhances the speed of tumor growth. Comparedwith the control group 1 (vehicle) the mean tumor size started to differsignificantly starting with day 45 until the end of the study. Noenhanced tumor growth was observed after end of treatment (day 68).

Animals of group 3 (5 mg/kg b.w. hIgG1) revealed a marked decrease intumor growth in comparison to group 1 (vehicle), getting statisticallysignificant with day 38 until day 83. The mean tumor volume started tostrongly regrow about two weeks after the end of treatment. One out often tumors disappeared at day 45 and did not regrow up to 19 days afterend of treatment.

The best performance of all treatment groups starting with 92 mm³ tumorvolume was found in group 4 (5 mg/kg b.w. muIgG2a), where the mean tumorvolume showed clear regression and tumors even disappeared in 4 animalsuntil the end of the observation period. The difference to the meantumor volume of group 1 (vehicle) was highly significant beginning fromday 38 until the end of study.

The early treatment with 1 mg/kg b.w. hIgG1 between days 14 and 36(group 5) revealed an early as well as long lasting effect on tumordevelopment. One animal has been excluded from further analysis as thetumor grew into muscular tissue. On day 31, only five animals had ameasurable tumor at the site of inoculation, in comparison to the restof the inoculated animals, where only 2 out of 60 did not respond totumor inoculation. The tumor progression was delayed of about 31 days(comparison of day 52 of control group 1 with day 83 of group 5). About50% of the animals did not show tumors at the site of inoculation at theend of the study.

2.7 Conclusion

No specific tumor or substance related clinical findings or mortalitywere observed in comparison with group1 (control).

No drug related interference with weight development was observed.

Tumor growth of RPMI8226 tumor cells after treatment was reduced in theorder of efficiency: hIgG1 1 mg/kg, 14-36 days every second day (group5)>muIgG2a 5 mg/kg 32-68 days every second day (group 4)>hIgG1 5 mg/kg32-68 days every second day (group 3)>hIgG1 1 mg/kg 32-68 days everysecond day (group 2). In groups 2 to 4, mean tumor volumes were againincreased after end of treatment to varying extents.

REFERENCES

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1-114. (canceled)
 115. An isolated monoclonal non-chimeric, non-CDRgrafted antibody based on a human sequence that binds human CD38 (SEQ IDNO: 22), (a) wherein said antibody mediates killing of a CD38+ targetcell by antibody dependent cellular cytotoxicity with an EC50 of 1 nM orless when a human PBMC cell is employed as the effector cell, whereinsaid CD38+ target cell is selected from LP-1 (DSMZ: ACC41) or RPMI-8226(ATCC: CCL-155), and wherein the ratio of effector cells to target cellsis from 30:1 to 50:1, (b) wherein said antibody mediates killing of aCD38-transfected CHO cell by complement dependent cytotoxicity with anEC50 of 3.2 nM or less, (c) wherein said antibody comprises a VH3variable heavy chain and kappa variable light chain, and (d) wherein theantibody comprises an IgG1 constant region.
 116. The antibody accordingto claim 115, wherein the variable heavy chain comprises a framework 1region comprising SGGGLVQPGGSLRLSC, a framework 2 region comprisingVRQAPGKGLEW, a framework 3 region comprising FTISRDNSKNTLYLQMNSLRAEDTAVand a framework 4 region comprising GQGTLVTV.
 117. The antibodyaccording to claim 116, wherein the variable light chain comprises aframework 1 region comprising TQSP, a framework 2 region comprisingQKPG, a framework 3 region comprising RFSGSGSGTDFTL and a framework 4region comprising TFGQGTKVEIK.
 118. The antibody according to claim 115,wherein the antibody mediates killing of a CD38+ target cell by antibodydependent cellular cytotoxicity with an EC50 of 0.60 nM or less. 119.The antibody according to claim 118, wherein the antibody mediateskilling of a CD38+ target cell by antibody dependent cellularcytotoxicity with an EC50 of 0.28 nM or less.
 120. The antibodyaccording to claim 119, wherein the antibody mediates killing of a CD38+target cell by antibody dependent cellular cytotoxicity with an EC50 of0.17 nM or less.
 121. The antibody according to claim 115, wherein theantibody mediates killing of a CD38-transfected CHO cell by complementdependent cytotoxicity with an EC50 of 0.94 nM or less.
 122. Theantibody according to claim 121, wherein the antibody mediates killingof a CD38-transfected CHO cell by complement dependent cytotoxicity withan EC50 of 0.41 nM or less.
 123. The antibody according to claim 115,wherein the antibody mediates killing of a CD38-transfected CHO cell bycomplement dependent cytotoxicity with an EC50 in the range of 3.2 nM to0.94 nM.
 124. The antibody according to claim 115, wherein the antibodymediates killing of a CD38-transfected CHO cell by complement dependentcytotoxicity with an EC50 in the range of 3.2 nM to 0.41 nM.
 125. Theantibody according to claim 115, wherein the antibody has a CDRH1 havinga length about 5, CDRH2 having a length about 17, CDRH3 having a lengthabout 13, a CDRL1 having a length about 11, CDRL2 having a length about7, and CDRL3 having a length about
 9. 126. The antibody according toclaim 115, wherein said antibody binds an epitope of CD38 that containsone or more amino acid residues within amino acids 44-66 of CD38 (SEQ IDNO: 22).
 127. The antibody according to claim 115, wherein said antibodybinds an epitope of CD38 that contains one or more amino acids residueswithin amino acids 110-122 of CD38 (SEQ ID NO: 22).
 128. The antibodyaccording to claim 115, wherein said antibody binds an epitope of CD38that contains one or more amino acids residues within amino acids148-164 of CD38 (SEQ ID NO: 22).
 129. The antibody according to claim115, wherein said antibody binds an epitope of CD38 that contains one ormore amino acids residues within amino acids 186-200 of CD38 (SEQ ID NO:22).
 130. The antibody according to claim 115, wherein said antibodybinds an epitope of CD38 that contains one or more amino acids residueswithin amino acids 202-224 of CD38 (SEQ ID NO: 22).
 131. The antibodyaccording to claim 115, wherein said antibody competes in binding toCD38 with an antibody comprising an H-CDR1, H-CDR2 and H-CDR3 depictedin SEQ ID NO: 5 and an L-CDR1, L-CDR2 and L-CDR3 depicted in SEQ IDNO:13.
 132. The antibody according to claim 131, wherein said antibodybinds to the same epitope of CD38 of an antibody comprising an H-CDR1,H-CDR2 and H-CDR3 depicted in SEQ ID NO: 5 and an L-CDR1, L-CDR2 andL-CDR3 depicted in SEQ ID NO:
 13. 133. The antibody according to claim115, wherein said antibody binds an epitope of CD38 that contains one ormore amino acids residues within amino acids 192-206 of CD38 (SEQ ID NO:22).
 134. The antibody according to claim 115, wherein said antibodycompetes in binding to CD38 with an antibody comprising an H-CDR1,H-CDR2 and H-CDR3 depicted in SEQ ID NO: 6 and an L-CDR1, L-CDR2 andL-CDR3 depicted in SEQ ID NO:
 14. 135. The antibody according to claim134, wherein said antibody binds to the same epitope of CD38 with anantibody comprising an H-CDR1, H-CDR2 and H-CDR3 depicted in SEQ ID NO:6 and an L-CDR1, L-CDR2 and L-CDR3 depicted in SEQ ID NO:
 14. 136. Theantibody according to claim 115, wherein said antibody binds an epitopeof CD38 that contains one or more amino acids residues within aminoacids 82-94 of CD38 (SEQ ID NO: 22).
 137. The antibody according toclaim 115, wherein said antibody binds an epitope of CD38 that containsone or more amino acids residues within amino acids 158-170 of CD38 (SEQID NO: 22).
 138. The antibody according to claim 115, wherein saidantibody competes in binding to CD38 with an antibody comprising anH-CDR1, H-CDR2 and H-CDR3 depicted in SEQ ID NO: 7 and an L-CDR1, L-CDR2and L-CDR3 depicted in SEQ ID NO:
 15. 139. The antibody according toclaim 138, wherein said antibody binds to the same epitope of CD38 withan antibody comprising an H-CDR1, H-CDR2 and H-CDR3 depicted in SEQ IDNO: 7 and an L-CDR1, L-CDR2 and L-CDR3 depicted in SEQ ID NO:
 15. 140.The antibody according to claim 115, wherein said antibody binds anepitope of CD38 that contains one or more amino acids residues withinamino acids 82-94 of CD38 (SEQ ID NO: 22).
 141. The antibody accordingto claim 115, wherein said antibody binds an epitope of CD38 thatcontains one or more amino acids residues within amino acids 142-154 ofCD38 (SEQ ID NO: 22).
 142. The antibody according to claim 115, whereinsaid antibody binds an epitope of CD38 that contains one or more aminoacids residues within amino acids 158-170 of CD38 (SEQ ID NO: 22). 143.The antibody according to claim 115, wherein said antibody binds anepitope of CD38 that contains one or more amino acids residues withinamino acids 188-200 of CD38 (SEQ ID NO: 22).
 144. The antibody accordingto claim 115, wherein said antibody binds an epitope of CD38 thatcontains one or more amino acids residues within amino acids 280-296 ofCD38 (SEQ ID NO: 22).